Morphological changes ofHelicobacter pylori after antibacterial therapy: an electron microscope study

To investigate the effect of morphological change ofHelicobacter pylori on gastric mucosal inflammation,H. pylori was examined by means of an electron microscope and computer-assisted three-dimensional reconstruction before and after antibacterial therapy. The results showed that the bacterium was usually present in different forms and sizes. Most of these variations are helicobacter shaped, 3–5 μm long (43%), or campylobacter shaped, 1–3 μm long (38%); fewer are round or coccoid (19%), and only 0.5–1 μm long. Active inflammation and an abnormal amount of mucus in the epithelial cells are the main changes seen in gastric mucosa withH. pylori infection. In some cases, 6 weeks after antibacterial therapy,H. pylori could still be observed by toludine blue, with slight positive results withH. pylori-antibody-labeled immunostaining. In contrast to the bacteria before antibacterial therapy, in mostH. pylori-positive cases usually only a minute number of bacteria were seen in coccoid form. These surviving coccoid-form bacteria showed thickened walls, and the surrounding periplasmic space was reduced or nonexistent. The epithelial cells were almost normal and¹³C-text negative. Although neutrophil infiltration was seldom seen, there were still some surviving bulblike cells with excess amounts of mucus in the gastric pits. It is suggested that the coccoid form ofH. pylori may be a static bacterium without urease production. For therapeutic decisions, it may be more important to identify whether there is an active inflammation in the gastric mucosa than to detectH. pylori.     

Eradication of Helicobacter pyloriin clinical situations

Abstract. Helicobacter pylori is prevalent worldwide, especially in developing countries, and is associated with several upper gastrointestinal diseases. Since it is present in over 90% of duodenal ulcer patients, empirical eradication in these patients is often recommended. In gastric ulcer patients, eradication is indicated only after the infection is confirmed. Testing for H. pylori infection should be carried out in patients with peptic ulcer hemorrhage, because eradication has been shown to reduce recurrent bleeding. Both H. pylori and NSAIDs are risk factors for peptic ulceration, and it is reasonable to screen for and eradicate H. pylori infection in peptic ulcer patients taking NSAIDs. H. pylori is a group I carcinogen for gastric adenocarcinoma, and should be eradicated for the primary prevention of this cancer. Eradication of this organism has been reported to result in regression of early low-grade mucosa-associated lymphoid tissue lymphoma. The role of H. pylori infection in the causation of gastroesophageal reflux and non-ulcer dyspepsia is not clearly established. Several tests are available for the diagnosis of H. pylori infection. These include invasive tests, such as histology, culture and urease test, and non-invasive tests, such as serology, urea breath test and stool antigen test. The choice of test is determined by clinical indication, pretest probability of infection, as well as the availability, cost, sensitivity and specificity of the test. H. pylori eradication therapy using proton pump inhibitor with clarithromycin and amoxycillin for 7 days has a success rate of 85–90%. Improved living standard and sanitation are vital in the control of H. pylori transmission and infection. Future development may include the use of vaccines against H. pylori, and therapies specifically targeting cagA strains of the bacteria.     

Interleukin-6 polymorphism and Helicobacter pylori infection in Brazilian adult patients with chronic gastritis

Abstract Helicobacter pylori is recognised as the most common cause of chronic active gastritis and this bacterium is also an important pathogenic factor in peptic ulcer disease. The biological factors that influence clinical outcome in H. pylori infection have been extensively studied. In addition to immunological factors in the host, bacterial virulence determinants in H. pylori strains are likely to play a crucial role in gastric cancer development. Singlenucleotide polymorphisms at the 5' flanking region of the interleukin (IL)-6 gene promoter (G or C at -174 base) have been identified and individuals with the G allele at position -174 have been shown to produce higher levels of IL-6 than those with the C/C genotype. The mucosal levels of IL-6 were reported to be increased in H. pylori-associated gastritis. The present study was conducted to examine any relationship between inflammatory cytokine polymorphisms and the inflammatory process in mucosa infected by H. pylori. In our study we did not find any association between the C and G alleles in adult patients with chronic gastritis and inflammatory process in gastric mucosa.     

Host cell signaling in Helicobacter pylori infection

Helicobacter pylori represents a highly successful human microbial pathogen that has infected approximately half of the world's population. This gram-negative microorganism colonizes the human epithelial layer in the stomach and induces a state of chronic inflammation that does not resolve the underlying infection and often leads to gastric or duodenal ulcers, or more rarely to gastric cancer. Among the reactions in H. pylori-infected epithelial cells the induction of proinflammatory cytokines, cell spreading and movement, as well as a scattered phenotype appear strictly dependent on the expression of pathogenicity island-encoded proteins in H. pylori. This review will discuss the features of the H. pylori-induced signal transduction leading to changes in host cellular function. Topics discussed comprise the signaling and the phenotypes associated with the type IV secretion system, the activation of target genes involved in gastric physiology, and putative mechanisms leading to the development of gastric cancer.     

Helicobacter pylori and cagA and vacA gene status in children from Brazil with chronic gastritis

Abstract. Helicobacter pylori has been shown to be strongly associated with chronic gastritis, gastric and duodenal ulceration, and is a risk factor for gastric carcinoma. Histology, urease, culture, and polymerase chain reaction have been employed as for H. pylori diagnostic methods, pre and post treatment or during follow-up of dyspeptic adult individuals referred for endoscopy. In order to obtain a more-sensitive and specific method for H. pylori detection, we evaluated gastric body and antrum biopsies of 134 consecutive Brazilian consecutive dyspeptic children aged 1–16 years by rapid urease test, histology and polymerase chain reaction using two pairs of oligonucleotides. Our results indicated that polymerase chain reaction with Southern blotting and hybridization with specific chemiluminescent probes increased the number of positive H. pylori patients by 35%. The genotyping of H. pylori strains directly from gastric biopsy using the same nucleic acid methodology revealed that there is no association of chronic gastritis in our infant patients with vacA s1 and the presence of the cagA gene. These data suggest an initial infection of children with normal mucosa and probably others factors than vacA s1 genotype or the presence of the cagA gene are associated with the onset of gastric disease. Altogether, our results reinforce the need for using more sensitive diagnostic methods in order to understand the role of H. pylori in the genesis of gastric disease in children and its progression in adults.     

Lipopolysaccharide in bacterial chronic infection: Insights from Helicobacter pylori lipopolysaccharide and lipid A

Lipopolysaccharides are generally considered toxic components of the Gram-negative bacterial outer membrane with potent immunomodulating and immunostimulating properties, but their contribution to adaptation of a given bacterial species to its microbial niche is, however, predominantly overlooked. Helicobacter pylori, as a cause of long-term infection in the gastroduodenal tract, has been proposed as a model for investigating and understanding the dynamics of bacterial persistence and parasitism in chronic infections. This review examines the structure and properties of H. pylori lipopolysaccharide and its lipid A moiety, and the insights that have been gained into their contribution to chronic infection and pathogenesis, including evasion and dampening of innate immune responses.     

MUC 1 mucin content in gastric juice of duodenal ulcer patients: effect of <Emphasis Type="Italic">Helicobacter pylori</Emphasis> eradication therapy

It has been suggested that Helicobacter pylori can interact via carbohydrate structures with gastric mucins. Particularly, the Lewis b structures of the secretory MUC 5AC mucin are considered to be putative receptors for bacterial adhesins. Also the epithelial MUC 1 mucin is implicated by some authors to have a major role in the mechanism of infection. The main objective of our study was to evaluate MUC 1 mucin levels in human gastric juice before and at the end of eradication therapy. Any possible changes could suggest the participation of MUC 1 in H. pylori infection. We assume that the amount of the soluble form of MUC 1 exfoliated to the juice correlates with MUC 1 expressed on epithelial cells. Gastric juice samples of 14 duodenal ulcer patients infected with H. pylori were assayed before and at the end of eradication. In all samples, DNA content was determined. Mucin fractions were isolated by gel exclusion chromatography. High molecular mass material containing MUC 1 was subjected to 4%–12% polyacrylamide gradient gels, electrotransfer to Immobilon P and immunodetection. In 12 infected patients (86%), the initial low level of MUC 1 mucin in gastric juice increased at the end of eradication. In comparison to the infected patients, neutral carbohydrate and DNA content in gastric juice diminished after treatment. Our results indicate that the bacterium affects the soluble form of MUC 1 mucin, thus suggesting a likely role of this mucin in the course of H. pylori infection.     

Localization of neurofilament protein mRNA in rat trigeminal ganglion byin situ hybridization at light and electron microscopic levels

By use ofin situ hybridization with a³⁵S-labeled oligonucleotide probe, the mRNA for neurofilament protein was localized to the trigeminal ganglion cells of rats. The result was visualized with radioautography at both light and electron microscopic levels. For the latter purpose, the cryostat sections were treated forin situ hybridization prior to embedding in epoxy resin. The presence or absence of detergent in the hybridization solution proved to be critical for the electron-microscopicin situ hybridization in terms of the signal/background ratio and ultrastructural integrity.     

Ontogeny of the endocrine cells of the stomach of sea bass (Dicentrarchus labrax L.): an ultrastructural study

The endocrine cells present in the developing stomach of sea bass larvae have been characterized ultrastructurally. Only one endocrine cell type (type I) was found in the presumptive stomach of 9- and 12-day-old larvae, one (type II) and five (types III, IV, V, VI and VII) in the aglandular stomach of 32-, and of 39- to 46-day-old larvae, respectively, and five (types III, VIII, IX, X and XI) in the differentiated stomach of 55- and 60-day-old larvae. A maturation process was established for some of these cells. Types I, II and III and types IV and X were thought to be different maturational stages of the same endocrine cell type.     

Ontogeny of the endocrine cells of the intestine and rectum of sea bass (Dicentrarchus labrax L.): an ultrastructural study

Several endocrine cell types were ultrastructurally characterized during the differentiation of the intestine and rectum of sea bass (Dicentrarchus labrax L.) larvae. Only one cell type (type I) was found in the posterior region of the undifferentiated gut of 5-day-old larvae (phase I). Types V and VI were found in both the intestine and rectum, types II, III and IV in the intestine, and types VII and VIII in the rectum of 9- and 12-day-old larvae (phase II), the rectum alone showing signs of functional differentiation. In phase III larvae, in which both the intestine and rectum were differentiated, types IX, X, XI, XII, XIII, XIV and XV were found in the intestine, only types X, XI and XII being seen in the rectum. Besides these, a new cell type, XVI, was observed in the intestine of 55- and 60-day-old larvae (phase IV), in which the digestive tract was completely differentiated. The endocrine cells appearing in phases I and II showed very scarce secretory granules and the ultrastructural features of undifferentiated cells. Some endocrine cell types in the earliest developmental stages were related to some of those found later. A maturational process of the endocrine cell types paralleled the differentiation of the intestine and rectum, with an apparent increase in the number of secretory granules accompanying organelle development.     

Choice of an adequate promoter for efficient complementation in Saccharomyces cerevisiae: a case study

Conservation of the function of open reading frames recently identified in fungal genome projects can be assessed by complementation of deletion mutants of putative Saccharomyces cerevisiae orthologs. A parallel complementation assay expressing the homologous wild type S. cerevisiae gene is generally performed as a positive control. However, we and others have found that failure of complementation can occur in this case. We investigated the specific cases of S. cerevisiae TBF1 and TIM54 essential genes. Heterologous complementation with Candida glabrata TBF1 or TIM54 gene was successful using the constitutive promoters TDH3 and TEF. In contrast, homologous complementation with S. cerevisiae TBF1 or TIM54 genes failed using these promoters, and was successful only using the natural promoters of these genes. The reduced growth rate of S. cerevisiae complemented with C. glabrata TBF1 or TIM54 suggested a diminished functionality of the heterologous proteins compared to the homologous proteins. The requirement of the homologous gene for the natural promoter was alleviated for TBF1 when complementation was assayed in the absence of sporulation and germination, and for TIM54 when two regions of the protein presumably responsible for a unique translocation pathway of the TIM54 protein into the mitochondrial membrane were deleted. Our results demonstrate that the use of different promoters may prove necessary to obtain successful complementation, with use of the natural promoter being the best approach for homologous complementation.     

Cerebral malaria in the rhesus monkey (Macaca mulatta): Light and electron microscopic changes in blood cells and cerebrovascular endothelia

To assess the interaction between the cellular elements of the blood and neurovascular endothelia in cerebral malaria, brain tissue from adult rhesus moneys (Macaca mulatta) infected with a virulent (W1) strain of Plasmodium knowlesi were studied by light and electron microscopical techniques. Light microscopical examination showed sequestration of macrophages and margination of erythrocytes containing late stages of the parasite in the capillaries and venules throughout the brains of the infected monkeys. Brain microvascular lesions (associated with parasitised erythrocytes and macrophage attachment to vascular walls) seen with the electron microscope, were swelling of the endothelial cells, formation of pseudopodia, increased numbers of pinocytotic vesicles and disorganisation of the mitochondria. Parasitised mature erythrocytes and macrophages adhered to the vascular endothelial lining in equal proportions. The endothelial ultrastructural alterations were similar to those described in experimental rodent and in clinical human cerebral malaria.     

Development of the mauthner cell in Xenopus laevis: A light and electron microscopic study of the perikaryon

Summary The Mauthner cell can first be recognized in Xenopus embryos at stage 31⁺–32 by its position and by the size of its nucleus and nucleolus. At early stages the perikaryon is dominated by a large ellipsoidal nucleus containing a single relatively compact nucleolus. The cytoplasm is characterized by numerous free ribosomes, poorly developed membrane systems, yolk platelets and globules, and by the presence of dense bodies containing what appear to be remnants of yolk breakdown.During subsequent development, membranes of both the endoplasmic reticulum and the Golgi apparatus become increasingly numerous. Long sinuous tubules of the endoplasmic reticulum are replaced by shorter repeatedly branching cisterns, and incidents of fusion between these elements and the nuclear envelope are less frequent. Large intramitochondrial granules and yolk elements are seldom encountered. The number of multivesicular bodies, alveolate vesicles and small secondary lysosomes increases. The originally nearly homogeneous cytoplasm is rearranged into areas rich in ribosomes and membranous organelles separated by channels containing predominantly neurofilaments and microtubules. At intermediate stages of development a cytoplasmic inclusion consisting of short arcs of parallel beaded strands, each 130–150 Å in diameter, is often present. The relationship of these morphological changes to the overall synthetic pattern of the cell is discussed. It is suggested that they signal the end of seeking growth by the cell.Supported by U.S. Public Health Service Training Grants GM 406 and NS 05591.     

Spatial growth and pattern formation in the small intestine microvascular bed from larval to adult Xenopus laevis: a scanning electron microscope study of microvascular corrosion casts

The microvascular anatomy of the small intestine of metamorphosing tadpoles of the South African Clawed Toad, Xenopus laevis (Daudin) is studied from developmental stages 55 to 65 and in adults by scanning electron microscopy (SEM) of vascular corrosion casts (VCCs) and light microscopy. Up to stage 62, VCCs reveal a dense two-dimensional vascular network ensheating the intestinal tube, whose proximal portion forms a clockwise spiralling outer and its distal portion an anti-clockwise spiralling inner coil. Vessels of the intestinal network impose flat and run circularly to slightly obliquely. Locally, dense capillary plexus with small “holes” indicating ongoing intussusceptive microvascular growth (IMG) and vessel maturation, are present. The typhlosole, an invagination along the proximal portion of the small intestine, reveals a dense capillary bed with locally ongoing IMG. VCCs of stages 62/63 for the first time reveal a three-dimensional vascular bed with longitudinal intestinal folds of varying size and heights greatly enlarging the luminal exchange area of the intestinal tube. From stage 65 onwards, longitudinal intestinal folds undulate and, though smaller in size and less mature as indicated in VCCs by the presence of wider, sinus-like vessels with small “holes” interposed between, closely resemble the intestinal folds present in the small intestine of adult Xenopus. Our data suggest that maturation of the vascular pattern in the small intestine of X. laevis tadpoles takes place successively after stages 62–63, and growth during this period is preferentially by intussusception.     

Nitric oxide contributes to both spinal nociceptive transmission and its descending inhibition in rats: an immunocytochemical study

The present study was designed to determine if nitric oxide (NO) was involved both in the dorsal horn responses to the primary nociceptive inputs and the descending inhibitory action on these responses. The first part of the experiments showed that when formalin was injected into one hindpaw, the nociceptive c-fos expression in the lumbar dorsal horn ipsilateral to the injection was suppressed dose-dependently by intrathecal (i.t.) administration of N^ω-nitro-l-arginine (l-NNA), a nitric oxide synthase inhibitor. In the second part of the study, the formalin injection was carried out into two hindpaws of the rats with a sectioned dorsal quadrant at the thoracic spinal level, in these rats, there was a significant suppression of c-fos expression in the dorsal horn on the side with intact dorsal quadrant, reasonably owing to the preservation of the spinally descending inhibitory fibers from the supraspinal level; furthermore, this suppression could be canceled following i.t. l-NNA administration. The results suggest that endogenous NO not only facilitates the perception of nociceptive inputs at the spinal level but also enhances the descending inhibition upon the spinal nociception.